Hydrophobic Affinity Chromatography of Proteins · Hydrophobic Affinity Chromatography of Proteins...

ANALYTICAL BIOCHEMISTRY 52, 430-448 (1973)

Hydrophobic Affinity Chromatography of Proteins

Received Junr 26, 1972; accepted November 10. 1972

1. Chromatographic studies have been made of the affinities of a number of purified proteins for agarose (Sepharose 4B) substituted with 4-phenylbutylamine (PBA) or with E-aminocaproyl-n-tryptophan methyl ester (ACTME) as compared to controls of untreated agarose and/or of cyanogen bromide treated agarose without addition of a substituting amine.

2. At pH 8, cu-chymotrypsin (3.4.4.5) and 7s y-globulin bind virtually

“irreversibly” to agarose-PBA, even in the presence of 1 M NaCl. Serum albumin, ,8-lactoglobulin. and ovalbumin also bind strongly to agarose-PBA at pH 8 and -0.05 ionic strength but are to different extents elutrd 1)~ 1 M NaCl. In each case elution by salt is enhanced by the presencr of a polarity reducing agent, e.g.. ethylene glycol. The findings suggest that binding of these proteins to agarose-PBA results from the combined (and possibly mutually reinforcing) effects of hydrophobic and rlrcl rost::tic forces.

3. Of t,he above proteins cY-chymotrypsin and y-globulin exhibit the highest affinity for agarose-ACTME (as well as for agarose-PBB). Serum albumin displays strong affinity at pH 5. These proteins are to a large extent eluted from agarose-ACTME by 1 ELI NaCl, in contrast to the KW of agarose-PBA. However. as with agarose-PBA, relcasc from binding is greatly enhanced by the added presence of a polarity reducing agent.

4. The applicability of hydrophobic affinity to protrin separations is demonstrated by chromatography of a protein mixture on an agarose-PBA column. Furthermore, several esteroproteolytic enzymes in crude prcpara- tions were separated from the bulk of the protrin and from each other b> rhromatography on agarose-BCTME.

Hydrophobic bonding’ may be the most important single factor in noncovalent interactions in aqueous solutions where the strengths of electrostatic, charge transfer, and hydrogen bonds are reduced by the

‘With the technical assistance of S. Frank Otillio. ‘The definition of “hydrophobic bonding” which is employed is that of Jemrlta

(1) : “an interaction of molecules with rach other which is stronger than the interaction of the separate molerules with water and which cannot be accounted for by covalent, electrostatic, hydrogen bond, or charge transfer forcsrs.”

430 Copyright @ 1973 by Academic Press, Inc. ill1 rights of reproduction in any form reserved.

HYDROPHOBIC AFFIMTY CHROMATOGRAPHY 431

charge solvating and hydrogen-bonding ability of water (,I). For in- stance, hydrophobic forces play an important role in the formation of the three dimensional native structure of proteins (2). In the case of chymotrypsinogen A, it has been shown (3) that neutralization t,hrough carbamylation of all or most of the 15 primary amino groups has little or no effect on the reformation of the biologically active zymogenic structure from the urea-denatured protein. This suggests that, at least in this case, electrostatic and possible other polar forces play a negligible role.

Although hydrophobic groups tend to be located in the interior of a protein molecule and thus would be inaccessible, early investigations (4) showed that hydrophobic effects are important in the interaction of certain enzymes with substrates and modifiers, indicating the accessibility of hydrophobic sites. The wide occurrence of often “functional” hydrophobic binding sites, which has been recognized only relatively recently, appears to involve not only many different types of enzymes (5,6) but also a disparate array of other proteins such as immuno- globulins (7,8), serum albumin, ,8-lactoglobulin, and ovalbumin (9).

Studies in aqueous solution of the interactions of proteins with hydrophobic ligands often are hampered by the low solubility of the latter. Soluhilization of such ligands by the addition of polarity-reducing agents to the medium can be expected to not only reduce their inter- act’ion with hydrophobic sites of the protein but also may affect the stability of the protein structure itself. However, as a means of achiev- ing molecular dispersion of an insoluble hydrophobic ligand (in order to study its interaction with proteins in an aqueous milieu) t,he ligand may be coralently attached to an insoluble hydrophilic matrix such as headed agarose. A major premise of the present investigation is that biologically active proteins in particular, which are often endowed with functional and accessible hydrophobic sites, may have varying degrees of affinity for chromatographic adsorbents of this type.

Hydrophobic effects presumably have been involved in many chro- mntogral)hic procedures including chromatography on the original “Tswett” columns. However, to our knowledge the possibility of using colurm chromatography for studying in aqueous milieu the affinity of immobilized hydrophobic ligands for hydrophobic functional sites in biologically actire proteins has not, previously been systematically investigated.”

The present communication is concerned with factors affecting the

’ ;\:o/e AdderI, in Proof: Th e manuscript of the present paper was submitted for publication brforc those of two recent articles on protein binding by v-alkylamino- agarows (31.32).

432 B. H. J. HOFSTEE

binding of various proteins to agarose substituted with 4-phenylbutylamine (PBAI using as controls both untreated agarose and CXBr- activated but unsuhstituted agarose (see below). In order to determine some of the factors that’ affect the interaction of certain proteins with the strongly hydrophobic phenyl- (CH,) q-residue, comparative studies have also been made with agarose substituted with the similar but more polar E-aminocaproyl-n-tryptophan methyl ester (ACTME) -1igand. Al- though the side chain of tryptophan also is strongly hydrophobic, the polar >NH and carbonyl groups provide for possible nonhydrophobic bonding with a protein. Both A-ACTME and A-PBA show strong affinity for a-chymotrypsin and similar enzymes ( 10,ll). They both are endowed with an interposing “arm” between the agarose matrix and the functional hydrophobic group. Such an arm has been shown to be of great importance for the attachment of certain enzymes to agarose-bound ligands (10,12,13).

The present results demonstrate that a variety of proteins, in adclition to chymotrypsin, display a high affinity for the -PBA as well as for the -ACTRIE ligancl. It is also shown that hydrophobic binding to an apolar ligand is affected by electrostatic or other polar forces, possibly involving the supporting matrix itself. The data generally suggest that “hydrophobic affinity chromatography” may prove to be a powerful new approach to the purification and study of proteins, at least. of those endowed with accessible hydrophobic sites.

EXPERIMENTAL

MatewIals

The purified protein preparations used were bovine serum albumin (Armour or CalBiochcm), p-lactoglobulin (N&r. Bioch. Corp.), ovalbumin, cY-chymotrypsin (3.4.4.5)) chymotrypsinogen il (Worthington) and 7S y-globulin (M arm). Pronase, a crude extract from Strepto- ?nyces griseus and containing several esteroproteolytic enzymes: was purchased from CalBiochem. Liver extracts were made from acetone powder prepared by the method of Horgan et al. (14). Tetraethyl- ammonium chloriclc was obtained from Eastman and was recrystallized from ethanol through the addition of ether. Ethylene glycol was “Baker analyzed.” Cyanogen bromide and PBA were purchased from Aldrich and dimethylformamide from Matheson, Coleman and Bell.

Unsubstituted agarose was used in the forms of Scpharose 4B (Phar- macia) or BioGel A 0.5 (Bio-Rad) ilgarose-~-aminocaproyl-o-tryptophan methyl ester was purchased from Ifiles-Yeda 1,td. Scpharose 4B

HYDROPHOBIC AFFINITY CHROMATOGRAPHY 433

substituted with 4-phenylbutylamine was prepared by the CNBr procedure described by Cuatrecasas (15j. Coupling of the activated poly- saccharide with PBA was carried out at pH 10-10.5. Solubilization of the amine was achieved by including 50 per cent tlimethylfornlamide in t,he reaction mixture (see also ref. llj. The final product, suspended in water and acidified to pH = 2 with HCI, was washed with water until the light absorbance of the filtrate at 260 mn was negligible. Its chymotrypsin-binding capacity in 0.05 nr Tris-HCl, pH 8 ( eeo below j , was ~1.5 mg per ml of settled slurry.

Agarose activated with CSBr, as described above, but without addi- t,ion of the amine is referred to as ‘iCA~Br-treuted” agnrose. This matc- rial was used as a conk01 in addition to untreated agarose.

The proteins were applied to columns of substituted agarose and to the control columns under identical conditions. When elution was carried out. with a concentration gradient,, the eluant was pumped continuously through the column by means of a peristaltic pump (Reeve Angel). Release of protein from t,he columns was continuously recorded employing either a Unicam double beam recording spectropho- tometer or a Turner fluorometer and a Dohrman recorder. For monitoring the activity of esteroproteolytic enzymes, an 0.01 M solution of ATEE’ or of BAEE was pumped into the eluate (after the latter had passed through t’he protein monitor) at a rate %-?/lo of the elution rate. The decrease in pH, measured with a Radiometer pH meter and continuously recorded, was taken as a measure of esteroproteolytic activity (16). For esterase(s) in crude liver acetone powder extracts, a saturated solution of ethylbutyrate in 0.01 M Tris-HCl, pH 8, was used as the sub- stratc. Further experimental details are described in the figure legends.

For a particular column, the retention volzl.r,Le of a solute (taken as a measure of its affinity for the adsorbent) is defined as its elution volume (at the point, when the solute is at’ its highest concentration in the flow-through cell of the monitor) minus the elution volume of a solute that is not retained (e.g., ovalbumin in the presence of 1 M SaCl, see below’) under otherwise identical conditions.

’ Thr nhhrcriations used are : ATEE, ncetyltyrosinc ethyl eater ; BAEE. bcnzoyl- arginine ethyl ester; ChT, a-chymotrypsin (3.4.4.5) ; ChT-ogen. chymotrypsinogen A; y-G, 7S r-globulin; BSA, ho\-ine serum albumin; OV. oralbumin ; p-LG, p-lactoglobulin; A-PBA and A-ACTME, agarose (Stpharose 4B) substiktd re- spectively with 4-phenylbutylsminc or c-nIniIlocnl,roq.l-n-tr?-ptophnn methyl ester; TEAC. tetr~etl~~lnmmonium chloride.


RESULTS

Binding of Variozwl P,roteins by Agarose Substituted with 4-PhenylblbtylaN1ine

Polar rind Apolar Effects on the Binding. As first shown by Stevenson and Landman (II), a-chymotrypsin is strongly bound by the A-PBA adsorbent. Figure 1 shows that the major portion of t,he enzyme remains adsorbed to the column during elution not only by the buffer (0.05 M

Trie-HCl, pH 8) but also after 1 M XaCl is added. On the other hand, it is readily released by 1 M tetraethylammonium chloride. As also shown in Fig. 1, untreated agarose fails to retain chymotrypsin under the same experimental conditions. Although the CNBr-treated agarose slightly retards the enzyme, the latter can be eluted with buffer in the absence of any additions.

The data of Fig. 2 indicate that binding of chymotrypsin also can bc reversed by a polarity-reducing compound such as ethylene glycol. Hoxvevcr, as can be seen, reversal of the binding to the hydrophobic ligancl is greatly enhanced in the presence of salt. The latter would be expected to increase rather than decrease hydrophobic binding (1). The findings indicate that binding is the result of a combination of apolar c_ and polar (e.g., electrostatic) forces (see below)

-- 70

h a- CHYMOTRYPSIN O.O5MTRIS-HCL, pH8

60 ‘3 Ii, g I ) UNTREATED g I ) AGAROSE $ I (C 1, 31 \ PBA- ‘;

.g ,’ RI AGAROSE I’, i

I:rc:. 1. Behavior of 1 mg of a-chymotrypsin on 1 ml (~0.5 X 5.5 cm) columns of untr(ialed. of CNBr-treated. or of PBA-substituted Sepharose 4B. The columns were ecluilibrated at room temperature with 0.05 M Tris-HCl buffer, pH 8. The loadrd I.olumns wvpre washed with several 2 ml portions of buffer alone followed by successive 2 ml portions of 1 M KaCl or 1 M tetraethylammonium chloride in huffrr. Two ml fractions per tube were collcctcd.

HYDROPHOBIC AFFIKITY CHROMATOGRAPHY 435

\GAROSE

t ,

TUBE NUMBER TUBE NUMBER

. I

20 25 30 35 40 3-o 35 40 TUBE NUMBER TUBE NUMBER

Fm. 2. Effect of 1 M NaCl on ethylene glycol elution of a-chymotrypsin from columns of PBA-substituted Sepharose 4B. The columns were equilibrated at room temperature with 0.05 M TrisHCl buffer, pH 8. washed 10X with 2 ml of buffer, and subsequently with 2 ml portions of the buffer in the presence of additives.

(A) 2.5 mg chymotrypsin was applied to a 5 ml (~1.1 X 5 cm) PBA-rolumn and eluted with 33% (v/v) ethylene glyrol in buffer in the absenre and in the presence of 1 M NaCl.

(B) 1 mg chymotrypsin applied to a 1 ml (~0.5 X 5.5 cm) column and eluted with 50% ethylene glycol in buffer in the absence and in the presence of 1 M NaCl.

7s X-GLOBULIN --1

FIG. 3. Behavior of l-2 mg of 7s y-globulin on 5 ml columns of untreated. CNBr-treated, or -PBA substituted Sepharose 4B. equilibrated at room temperature with 0.05 M Tris-HCl buffer, pH 7.5. The loaded columns were washed 12-18 times with 2 ml portions of buffer and then successively with several 2 ml portions of buffer containing either 1 M KaCl or 1 M NaCl plus 50% ethylene glycol.


The results of Fig. 3 show that in the absence of polarity reducing agents 7S y-ylob,ulin, like chymotrypsin, is strongly (virtually “irreversibly”,l bound to the A-PBA column. R,eversal of the binding of the major portion of the protein is achieved in the presence of a mixture of 50% ethylene glycol and 1 M NaCl. Like chymotrypsin (Fig. 1)) the protein has little or no affinity for untreated agarose. However, in contrast to the enzyme, y-globulin is rather strongly adsorbed on the control column of CNBr-treated agarose. Nevertheless, the behavior of the protein on the PBA- substit,utecl column differs ronsiderably from that, on the control column. As seen in Fig. 3, the protein is slowly leached from the latter by the buffer alone, whereas it is firmly bound to the PBA-column under the same conditions. Also, whereas ethylene glycol enhances elution of the protein from the PBA-column, ethylene glycol has little or no effect on binding of the protein to the CNBr- treated control column. This suggests that binding to the control column does not involve hydrophobic forces. It has been established that hydrophobic forces, often in cooperation with electrostatic forces, are involved in the interaction of antibodies u-it.11 antigens, haptens, or inhibitors (7,8)

In the absence of additions to the eluting buffer, the agarosc-PBA adsorbent strongly binds serum albumin whereas little or no binding is shown by the untreated and CNBr-treated agarosc controls (Fig. 4). Upon addition of 1 M NaCl to the eluting buffer, a portion of the adsorbed material is eluted from agarose-PBA, the remaining protein being elutecl by the further addition of ethylene glycol. These results suggest that, for this protein as well, both polar and apolar forces are involved in the binding. [Evidence has been presented (17) that 8 aeccssihle hydrophobic sites are present on the strum albumin molecule.] The data of Fig. 4 also indicate that the pH of the medium affects the relative contributions of the polar and apolar forces to the binding (see below j

The behavior of P-lactoylobulin on a column of A-PBA (Fig. 5) is similar to that of serum albumin (Fig. 41 in that a considerable port,ion of the protein is eluted by 1 M NaCl. However, the relative effect of added salt is larger and the effect of the subsequent addition of ethylene glycol is relatively smaller than for serum albumin, suggesting a lesser degree of hydrophobic interaction.

The results with ovalbumin (Fig. 5) also indicate strong binding to A-PBA as opposed to unsubstituted agarose. The binding, however, is readily and nearly completely reversed by the addition of 1 M NaCl. Only a small fraction remains bound under such conditions. Further data, in addition to those of Fig. 5, showed that ovalbumin also passes almost unretardedly through CNBr-treated agarose, but is virtually


UNTREATED AGAROSE

i

1. pH5

i ’ . pH8

CN Br -TREATED

,I ‘.

AGAROSE : pH5 . pH8

I I

15 20 25 30 35 TUBE NUMBER

FIG. 4. Behavior of 3 mg serum albumin on 5 ml columns of untreated, CNUr- treated and PBA-substituted Sepharose 4B at pH 5 (0.05 M acetate) and at pH 5 (0.05 M Tris-HCl) under otherwise t.he same conditions as for Fig. 3.

TUBE NUMBER

FIG. 5. Behavior of 3 mg of either ,8-lactoglobulin, ovalbumin, or chymotrypsinogen on 5 ml columns of untreated and of PBA-substituted Sepharosc 4B at pH 8 under otherwise the same conditions as for Fig. 3.


irreversibly absorbed by A-PBA unless the ionic strength is increased to -0.25 at which point it is rather abruptly and nearly completely released. In the presence of 33% ethylene glycol, the required increase in ionic strength is only ~0.1. In the absence of added salt, at least 50% ethylene glycol is needed for elution of the protein. Thus it appears that,, similarly to the above four proteins, ovalbumin binding to the agarose bound -PBA ligand depends on electrostat,ic and/or other polar effects and to a much larger degree (set Discussion).

Although of all the proteins tested, chymotrypsin displays one of the highest affinities for the agarose-PBA adsorbent, the affinity of chyrrzot~~~psinogen is the lowest. The latter protein displays a higher affinity for 9-PBA than for the control columns but can be eluted with 0.05 M Tris-HCl buffer in the absence of additions (Fig. 5). The diffcr- cnce in hydrophobic affinity between the enzyme and the zymogen is in accord with the proposition that the hydrophobic binding site in the enzyme active center is fully unmasked only after activation of the zymogen (l&19).

dppcrrent Inhomogeneity of the Protein with Respect to Hydra- phobic Binding. As seen from Figs. l-5, all of the proteins except chymotrypsinogen require additives to the eluant buffer in order to achieve reversal of binding to the A-PBA adsorbent. For most of these proteins, a fraction can be eluted upon addition of 1 M NaCI, whereas elution of the remainder of the protein requires the additional presence of a polarity reducing agent such as ethylene glycol. Whencrer tested, elution was incomplete or did not occur at all in the presence of relatively high concentrations of ethylene glycol alone, i.e., in the absence of added salt (e.g., Fig. 2).

For serum albumin (“electrophoretically l’ure” anal other highly purified preparat#ions) , the occurrence of separable fractions was confirmed by elution of t,he protein at 4°C in an cl.0 M NaCl gradient. One fraction was released when the NaCl concentration was about 0.3 M. The other fraction remained bound even in 1 M NaCl but could be elutcd when 50% ethylene glycol was also added to the eluant. These observations (Khich warrant further investigation) may indicate that two major protein conformations are involved in which the relative contributions of polar rind apolar forces to the binding to -PBA diffrr. On the other hand, inhomogeneity of the binding sites of the adsorbent, rather than inhomogcneity of the protein, cannot be excluded as yet.

Stnl)ility of the Proteins in the Eluant Solutions. Most, proteins appear to maint,ain their native structure in ethylene glycol at concentrations as high as 507, (20). For several of the present proteins, this is

HYDROPHOBIC AFFIKITB CHROMATOGRAPHY 439

ronfirmed by the following observations, which also indicate rtabilit) of these proteins in TEAC solutions under the experimental conditions.

ChT was fully active after treatment for 1.5 hr at room temperature in 50cJ1 iv/v 1 ethylene glycol solution in buffer followed 1);~ I.?fold dilut,ion into a reaction mixture containing 0.013 11 ~~r-llcl~to~los~-~~ei~- zonte as the substrate. It was also found that the enzyme is not inacti- vated in the preeencc~ of TEAC in 0.66 M concentration. The quaternary ammonium salt, in this concentration had no noticeable effect. on the absorbance of the enzyme at 292 nm. The latter decreases upon denaturation in 8 M urea (21). Thus it may be assumed that the enzyme is stable during clution with these solvents, particularly at low temperatures. Like ChT, ChT-ogen also shows the characteristic difference spectrum in the near uv range upon denaturation in 8 M urea. However, such a different spectrum was not observed by treatment of the protein with .50$~1 ethylene glycol at room temperature. Similar results, indicating the absence of denaturation under the experimental conditions, mere obt,ained with strum albumin and ovalbumin. However, the solubilitics of 7-G and of /?-LG were found too low for such studies.

Comparison of Ayarose-PBA and Aynrose-ACTME (IS Protein Adsodents

At relatively low ionic strength (20.01) and depending on pH, bincling of the above proteins to A-ACTME, as in the case of X-PBA, is usually much stronger than to unsubstituted agarose. However, in contrast to elution from the A-PBA adsorbent, the major portion of all of the proteins could he eluted from an A-ACTRIE column by the adclition of 1 M NaCl to the eluant, at least. at pH 8 (Table 1). On the other hand, in the cast of chymotrypsin (Fig. 6’1 and also for 7-G and BSh (not shouTn j, elution occurs much more readily by additionally reducing the polarit’y of the medium by using 1 M TEAC instead of KaCl, indicating that a liydropliot~ic effect is invoh-et1 in the binding of thcw proteins. This is further confirmed by the fact that most of the proteins that. are strongly bound by A--4CTME, are endowed with accessible hydrophobic groups. This is the caw for chymotrypsin ( l&19) and BSA (9,17j as well as for y-G (7,811. On the other hand, in addition to a more or less unspecific type of hydrophobic binding, a certain degree of specificity of the interaction bctncen protein and adsorbent can be expected even for proteins other than chymotrypsin lace Discussion). In any event, these observations, including those on the effects of ionic strengt,h and pH (Table l), indicate that, as in the care of -PBA (Figs. I-51, byclropl~obic as well ar electrostatic forces are involrctl in tlie


TABLE 1 The Effect of pH on the Retention of Proteins on Columns

of Agarose (Cont,rol) and of Agarose-ACTMEG

Agarose (conkol)

pH5 pH8 PH 5

Agarose-ACTME

Ah PH S A”

cu-Chymotrypsin s 11 > 100 >92 -75 -v64 Chymotrypsinogen A 8 !I 29 “1 27 18 Serum albumin 10 0 >lOO >90 4 4 Ovalhtrmin :: 0 13 10 3 3 p-Lart~oglobulin 5 0 21 16 0 0 7Sy-globulm 12 13 >lOO >XS 65 32

(1 The retention volumes (ml) given are for the major component of each of the purified protein preparations (l-2 mg). The columns were equilibrated at *3”C with either 10-a M acetate (pH 5) or 10-S M TrissHCl (pH X), the latter containing lo+ M CaCb. The proteins were ehtted by application of a linear NaCl gradient (O-l.0 M) to the buffer. The molarit,y of the salt gradient increased by abottt 0.01 M/ml of eluant. Columns were 0.5 X “l-24 cm.

*The effect on protein affinity of substitution of the agarose is indicated by the difference iA) in the retention volumes found for the substit’uted and unsubst,it)ut’ed (control) agarose adaorbents.

I- :, i l,,

CHYMOTRYPSIN

E ! \ ;‘?: PH8

Ij 1.

FIG. 6. Chromatograhy of 2 mg of a-chymotrypsin on -0.5 X 22 cm columns of agarose and of A-ACTME at -5’C. The columns mere equilibrated with 10m3~ Tris-HCl. pH 8. and eluted continuously with the buffer in the presence of a linear gradient. of NaCl or of N(C,H,),Cl (see Experimental and also legend to Table 1). The relative protein content of the rluate was monitored either by fluorescence (Fluor) or by absorbance (A,,,).

HYDROPHOBIC AFFINITY CHROMATOGR$PHY 441

binding of the proteins to A-ACTME. As could be expected, the hydrophobic forces generally are less predominant in the case of A-ACTME than for the more strictly hydrophobic -PBA ligand. However, the most important aspect of these findings would appear to be that, in addition to chymotrypsin (for the specific binding of which h-ACTME was “designed”), several other apparently unrelated proteins also are strongly bound by the adsorbent (WC Discussion/.

Analyses of Mixtures of Proteins by Chroumtogrnphy on

A-PBA or A-AC’TME

On the basis of the above results and as a demonstration of the poten- t’ialities and possible drawbacks of the present procedure for protein separation, a mixture of three purified proteins (chymotrypsinogen, ovalbumin, and serum albumin) was analyzed by chromatography on A-PBA (Fig. 7). With 0.5 mg of each protein, the entire mixture is adsorbed by 5 ml of the adsorbent. However, fraction 1 (presumably chymotrypsinogen, see Fig. 5) is slowly released by elution with the buffer in the absence of any additions. No further protein is released until the ionic strength of the eluant’ is raised to ~0.25 when, as was noted above, the major portion of ovalbumin is released from the column (fract.ion 2). As was shown by separate runs with serum albumin in a salt gradient8 (unpublishedI, part of this protein is released at NaCl concentrations somewhat higher than required for ovalbumin, the remainder not bcing released until 50% ethylene glycol is also added to the cluant (set also Fig. 4). These two serum albumin fractions may be assumed

Mtxture of CH T-ogen 0

CVAL B SER ALB

on FH2-AGARCSE

Frc:. 7. Sep3mtion of :f misturc of rhymotrypsinog~~n. ovnlbumin. and serum albumin (0.5 rnr each) by chromatography on a 5 ml PBA-substituted SPpharos: 4B colmnn :rt room trmperaturr. Elation was carried out continuously with 0.05 M Tris-HCl buffer. pH S. followd by a linear NaCl gradient (O-1.0 M) in buffer and finally with buffer containing 1.0 M KnCl plus 50% ethylene glycol. The protein concentration in the rluntc was continuously monitored by fluorcscrncc.

442 R. H. J. HOFSTEE

to be represented by fractions 3 and 4 of Fig. 7. Howl-ever, fraction 4 could also contain a minor portion of ovalbumin (see Fig. 5). Thus a particular adsorbent, although useful for the separation of certain proteins, may not be saGsfactory for the separation of others (see Discussion).

The results with a crude extract from pig liver acetone powder (,Fig. 8A) show that two ethylbutyrate-hydrolyzing enxymcs are separated from each other by chromatography on A-ACTME, one enzyme occur- ring in an eluate fraction of low protein content. Considerable separa- t,ion of the two active fractions is also obtained on an unsubstitutcd agarose column (Fig. 8B). However these active fractions coincide with the major protein fractions, i.e., little or no purification is obtained on the control column.

The results of chromatography of pronase on a column of A-hCTME (Fig. 9j indicate that one BAEE- and at least two ATEE-hydrolyzing fractions can be separated from the bulk of the protein and from each other. Although some separation is obtained on an unsubstitutcd control column, separation is much more effectively achieved on the A-ACTRIE column, as was the case with liver extract (Fig. 8i. Elu- tion from A-ACTME always occurred much more readily with TEAC than with NaCl. In contrast, the effects of TEAC and of NaCl on bind-

IA A-ACTME j

6~ LiVER EXTR

-I ~:fl

pH8, NaCl

20 40 60 80 RETENTION VOLUME (ML)

FIG. 8. Elution of 0.2 ml of an nc~ueoua (1: 10) eslracl of pig liver ac’vtom~ IIOU.C~I,I from an ACTME-s~lhstitut~d (A) and from an unsubstitutc~d agaroec (1~) c,olumn. Elut,ion was carried out at -5°C and pH 8 by means of n linear KaCl pradit,nt (see legend to Table I). The cluatc was monitored by ahsorbancc at 222 nm (A) or by fluorescence (B). after which a saturakd solution of ethyl hutyratc in lo-’ M TrisHCl, pH 8. was injected into the eluatc flow, and csterasr activity KW followed by continuous measurement of pH (see Experimental).

HYDROPHOBIC AFFINTT CHROMATOGRAPHY 443

-~~ -~ 100 120’

RETENTION VOLUME (ML)

FIG. 9. Continuous elution of 5 mg of pronase from a -0.5 X 20 cm column of 8-ACTME by means of an NnCl gradient at pH 8 at -5°C (see lrgend to Table 1). After monitoring the light nbsorbance (Ax?) of the eluate. either ATEE or BAEE was injected into the eluntc flow and the decrease in pH was recorded (we Experimental).

ing to unsubstituted agaroee were about the same. Therefore, binding to -ACThlE is at, least partially hydrophobic whereas binding to unsuhstituted agarose appears to be entirely through electrostatic or other polar forces.

DISCUSSION

Effects of Polwr Foxes on Hydrophobic Binding

The effects of pH and of ionic strength on the binding of various proteins to uncharged or cl-en to apolar ligands (Figs. 1-6, Table 1:) indi- cates that polar as well as apolar forces are involved. The possibility of conformational change in the protein upon changing the ionic conditions should be taken into considerat’ion in specific cases. It would be improbable, however, that all of the proteins of Table 1 undergo a conformational change upon changing the pH from 8 to 5, and that this change is favorable for binding and counteracted by the addition of salt. At, the lower pH, the possibility of increased hydrogen bonding involving the carbonyl group of -ACTME cannot be excluded, but this is not l’ossiblc for the -PBA ligand. It would seem mow likely that polar group on the supporting matrix itself, or possibly the matrix-bound nitrogen of the sub!&itutin g amine (see below), may play a role in the bincling.


Even highly purified agarose may carry negative charges (23). The presence of such charges is consistent with the present findings that at pH 8 and low ionic strength the more basic proteins (chymotrypsin and chymotrypsinogen) are bound by the unsubstituted agarose, wherc- as the acidic proteins (serum albumin, ovalbumin, and ,&lactoglobulin) which are negatively charged at pH 8, arc not (see Table 1). Thus the negative charges would attract, positively charged proteins towards the matrix and might thereby promote interaction (including hydrophobic bonding) with the substituting ligand. It would seem likely that such cooperation between (long range) electrostatic and (short range) hydrophobic forces might result in mutual reinforcement.

On the other hand, negatively charged proteins would be repelled by the negative charges on the matrix and binding to the substituting ligantl would diminish. Binding under such conditions would indicate that hydrophobic interactions are able to overcome electrostatic repulsion. It has been found for serum albumin (24) that the presence of a positive charge adjacent to a hydrophobic binding site increases binding of hydrophobic compounds that carry a negative charge, even when the overall charge on the protein is also negative. Such a positive charge adjacent to the hydrophobic binding site on the protein also could enhance interaction with a hydrophobic ligand bound to the supporting matrix in proximity to a negative charge. The addition of salt would tend to cancel this effect but, on the other hand, would simultaneously diminish the repulsive action between t’he negative charges of the matrix and the negat’ively charged protein molecule as a whole.

Another factor that could be involved here is the possible positive charge on the matrix bound amino nitrogen of the substituting ligand (13). Thus the overall charge of the adsorbent’ would be determined by the balance between negative charges on the matrix and positive charges introduced by the substit#uting amine. The combined effect of such charges, which would depend on their relative position, on the extent of substitution, and also on the particular protein under investigation, is difficult to assess.

For protein separation on the basis of hydrophobic interaction per se, it might seem expedient to simply quench electrostatic and other polar effects by including an appropriate amount of salt into the medium and then apply a gradient of a polarity reducing agent such as ethylene glycol. However, as can be seen from the data of Table 1, depending on the adsorbent employed and on the pH of the eluant, the salt (NaCl) concentration at which different proteins are released varies from 0 to >l M. The addition of an “excess” of salt would cancel any possible advantage of the charges in detecting small differences in affinity. Al-


though the A-PBA adsorbent, in 1 M NaCl and at pH 8, binds several of the proteins tested, A-ACTME under such conditions did not bind any of these proteins.

By applying a salt gradient rather than an excess of salt, the ljostu- latccl cooperative and mutually reinforcing effect of polar and apolar forces (which could magnify differences in hydrophobic binding and would vary from one protein to the next) would provide an adclitional parameter for protein separation. In this respect extremely strong liyclrol~l~obic binding i Y not necessarily an advantage. Bs was shown (Figs. 4 and 51, purified proteins often appear grossly inhomogeneous with respect to binding to A-PBA. Such inhomogeneity (which is an interesting subject for further study, is a drawback for protein separation by chromatography on columns of such an adsorbent (see Fig. 7 and pertaining text). Since this inhomogeneity did not occur or occurred t’o a much lesser extent, in the case of A-hCTME it appears related to the fact, that the esterified tryptophan residue of ,4-,4CTiUE is less hydrophobic than the phenyl residue of A-PBA. Furthermore, in several crude mixtures, including pronase and blood serum, a major fraction of the protein adhcrecl to A-PBA with such tenacity that regeneration of the adsorbent required treatment with organic solvents or even 8 M urea. On the other hand, binding to A-ACTME could largely be reversed merely by increasing the ionic strength of the medium. Similar considerations of the combined effects of hydrophobic and electrostatic forces have recently been discussed by Yen (25 1.

Specificity of Hydrophobic Internctions

Cuatrccasas et nl. (10) noted that at low ionic strength (e-0.01)) the A-ACTME adsorbent (which was “designed” for the specific adsorp- tion of chymotrypsin) also nonspecifically binds other proteins in which case binding is totally reversed by increasing the ionic strength. How- ever. the present results show (e.g., see Table 1) that the %onspecific” binding to A-ACTME often is much stronger than to unsubstituted agarose. This conclusion, which is based on relative values obtained under identical conditions, is not affected by recent obscrrations (26) t.hat the salt concentration needed to prevent binding may he lower than is required for release of the protein after the latter is concentrated on the column.

It is of interest that the -ACTRIE ligand, besides binding chymotrypsin, also strongly binds several other proteins: y-globulin and serum albumin (Table 1 ), one enzyme in liver extract (Fig. 8), in addition to at least three enzymes in pronase (Fig. 9), and also stem bromelain from crude extracts (27). A most likely common property of this dis-


parate array of proteins would be the accessibility of one or more hydrophobic sites. This is supported by the results with polarity rduc- ing agents such as TEAC and ethylene glycol and by the finding that generally the affinity of the proteins for the less polar PBA-ligand is even higher than for -ACTME.

Hydrophobic sites or “pockets” of enzymes often serve as points of attachment for ligands (19). Furthermore, this type of bonding may be an important factor in the formation of the antibody-antigen complex (78). The hydrophobic areas on the serum albumin molecule may be assumed to play a role in the binding and transportation of metabolites (and drugs) by this protein (28,29).

The present finding that a particular hydrophobic ligand may have a varying degree of affinity for the hydrophobic sites of many diffcrcnt proteins is in accord with the observations that the binding I)>- SCIYIII~

albumin of a great variety of entirely different compounds is s;rnply a function of their lyophilic character as measured by their octanol-water partition coefficients (30). On the other hand, a certain degree of specificity with respect to the size and shape of the interacting hydrophobic areas can be expected. For example, the sequence of decreasing affinity of the proteins of Table 1 for the -ACTME ligand, although similar, differs from the sequence of these same proteins with respect to hydrophobic binding to 2-p-toluidinyl naphthalene-6-sulfonate as obscrvecl by McClure and Edelman (9). Such differences could be related to the degree of “complementariness” or “fit” of the hydrophobic sites of various proteins with respect to a particular hydrophobic ligantl. Thus, in order to fully exploit differences in hydrophobic properties of different proteins, a series of adsorbents substituted with ligancls carrying different hydrophobic groups should be employed.

Chromatography on adsorbents of the above type would present an additional means for the separation and study of proteins and which could be used in conjunction with other procedures. Since the present procedure is based totally or partially on properties other than molecular size or electric charge, it would present opportunities for prot’ein separation and study not afforded by other means such as exclusion chromatography or procedures based on charge effects alone. Furthermore, because the hydrophobic ligands are relat#ively insoluble, their molecular dispersion through covalent binding to an insoluble but hydrophilic matrix, makes possible the study of t’heir interactions with biological materials in aqueous solutions in the absence of solubilizing agents. As contrasted to affinity chromatography on adsorbents designed to have specific affinity for a known biologically active protein, t’he present procedure would be more general in applicability. Because a particular

HYDROPHOBIC AFFIIiITY CHROMATOGRAPHY 447

hydrophobic adsorbent appears capable of binding a wide range of proteins, a relatively limited number of such adsorbents might suffice for the chromatographic separation and study of many proteins with accessible hydrophobic groups.

ACKXOWLEDGMEXTS

Thr ant hor acknowledges with great appreciation the hrll~ful ,srlggrslions 1):

Dr. Dolores Bobb in the prr1lnration of thr manuscript. This inwsligntion h:ta been supported by G. S. Public Hrall h Swvicr Gr:lnt No. FR-05513. the Hurw! Bassett Clarke Foundation, and by the S:mta Clara County I-nitrd Fund.

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